The electron-transfer protein adrenodoxin (ADX) binds to free cytochrome P450 (Kd equals 0.5 microns), but binding is greatly activated by the presence of cholesterol (kd equals 0.02 microns). The flavoprotein ADX-reductase binds ADX with even higher affinity, but a ternary complex of all three proteins does not form. Kinetics of cholesterol side chain cleavage indicate that ADX "shuttles" between ADX-reductase and P450 scc. Reaction is activated by increased by ionic strength but is inhibited by Ca ions (ID50 equals 50 microns). 11 beta-hydroxylation also utilizes an ADX-shuttle and is activated by increased ionic strength but in this case Ca ions is a potent activator. Salt may activate side-chain cleavage by enhancing the dissociation of inhibitory oxidized ADX, while for 11 beta-hydroxylation the salt effect is due solely to minimizing steady-state levels of oxidized ADX. A method has been developed for lysing adrenal mitochondria and separating inner and outer membranes, while minimizing cholesterol redistribution between the membranes. Aminoglutethimide, injected into stressed rats, effected an increase in cholesterol in the inner membrane via direct binding to P450scc. Cycloheximde enhanced total mitochondrial cholesterol and enhancing levels in the outer membrane. It is concluded that a rate-limiting event in ACTH-activation of adrenals is transfer of cholesterol from the outer mitochondrial membrane to the inner membrane and that this step is coupled to protein synthesis.